Analysis of the Molecular Mechanism and Physiological Role of Golgi Stack Formation and Golgi Biogenesis.

Abstract

The Golgi apparatus is a membrane-bound organelle that serves as a central conduit for the processing of membrane and secretory proteins in all eukaryotic cells. The Golgi comprises of stacks of flattened cisternae in most eukaryotic cells. Two homologous Golgi Reassembly Stacking Proteins, GRASP65 and GRASP55 mediate the formation of Golgi stacks in vitro. Using RNA interference and systematic electron microscopy, I showed that depletion of either GRASP55 or GRASP65 in vivo reduces the number of cisternae per stack, whereas simultaneous knockdown of both GRASPs leads to the disassembly of the entire Golgi stack, demonstrating complementary roles for GRASP55 and GRASP65 in Golgi stacking in vivo. Biochemical analysis reveals that GRASP55 and GRASP65 stack Golgi cisternae via a common mechanism by forming cell-cycle regulated oligomers. By depleting GRASP55 and GRASP65 with RNA interference, I demonstrated that Golgi unstacking accelerates protein trafficking, causes missorting of lysosomal protein cathepsin D, and alters the glycosylation of cell surface proteins. Subsequently, cell adhesion and migration are attenuated, possibly due to the reduction of cell adhesion molecules alpha5/beta1 integrins. Both bulk protein synthesis and cell proliferation in cells with unstacked Golgi are enhanced. These results suggest that Golgi cisternal stacking regulates protein transport and modifications, which are important for cell adhesion, migration and proliferation. In mammalian cells, Golgi biogenesis occurs through extensive disassembly at the onset of mitosis and subsequent reassembly in the two daughter cells. I demonstrated that mitotic fragmentation of Golgi is mediated via COPI vesicles formation, which requires the activity of ADP-ribosylation factor-1 (ARF1). Our data also suggest that the Golgi is an organelle of its own entity and inherits independently during mitosis. Previous studies indicate that mono-ubiquitination, which occurs in the disassembly process, is required for post-mitotic reassembly. We have identified a Golgi localized E3-ligase HACE1 and its potential substrate syntaxin-5, and thus provide information and tools to understand the role of ubiquitin in the regulation of Golgi dynamics during the cell cycle.